# Patent

## Title
Fusion genes associated with acute megakaryoblastoc leukemias

## Abstract

The invention relates to human nucleotide sequences which occur as a result of the t(1;22)(p13;q13) chromosomal translocation event which is known to occur almost invariable in young children with acute megakaryoblastic leukemia. The translocation results in the formation of fusion genes which encode fusion proteins. The invention provides the nucleotide sequences of transcripts of the fusion genes and the amino acid sequences of the fusion proteins encoded thereby. Also provided are methods for detecting the t(1, 22) translocation, for identifying agents capable of binding to the fusion protein and for identifying agents useful for treating patients with acute megakaryoblastic leukemia.

## Background
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This invention was made in part with U.S. Government support under Cancer Center Support (CORE) grants CA-21765 and CA-87064 from the National Cancer Institute. The Government may have certain rights in this invention.


FIELD OF THE INVENTION
The present invention is directed to the field of the molecular genetics of cancer. Specifically, the present invention relates to human acute megakaryoblastic leukemias in which a translocation between chromosomes 1 and 22 (referred to in the art as “t(1;22)”) has occurred. On a molecular level, the DNA rearrangement in t(1;22) results in two reciprocal fusion genes that each comprise segments of the RBM15 and MKL1 genes.
BACKGROUND OF THE INVENTION
Chromosomal abnormalities are frequently associated with malignant diseases. In a number of instances, specific chromosomal translocations have been characterized, which generate fusion genes encoding proteins with oncogenic properties (Sawyers et al., Cell 64:337-350 (1991)).
Recently, the cloning of chromosomal translocations has led to identification of pathogenically relevant oncogenic fusion transcripts and proteins in specific subsets of acute nonlymphocytic leukemia (ANLL), such as promyelocytic leukemia-retinoic acid receptor alpha fusion gene (PML-RARα) in acute promyelocytic leukemia (FAB-M3 subtype), the acute myeloid leukemia 1-eight twenty one fusion gene (AML1-ETO) in ANLL with maturation (FAB-M2), and various mixed lineage leukemia (MLL) fusions in acute myelomonocytic and monocytic leukemias (FAB-M4 and -M5) (Melnick, A. & Licht, J. D., Blood 93, 3167-3215 (1999); Downing, J. R., Br. J Haematol. 106, 296-308 (1999); Rowley, J. D., Semin. Hematol. 36, 59-72 (1999); Look, A. T., Science 278, 1059-1064 (1997); Faretta, M., Di Croce, L. & Pelicci, P. G., Sem. Hematol. 38, 42-53 (2001)). Despite these significant advances, little is known about the genetic mechanisms underlying acute leukemias of the megakaryoblastic (platelet precursor) lineage (AMKL, FAB-M7) (Cripe, L. D, infra). Almost invariably, AMKL in non-Down syndrome infants and young children harbor the t(1;22)(p13;q13) translocation, in most cases as the sole cytogenetic abnormality (Carroll, A. et al; Lion, T. et al., and Bernstein, J. et al., infra). Phenotypically, AMKL presents de novo (i.e., without a so-called preleukemic stage), with a large leukemia cell mass, and frequent fibrosis of bone marrow and other organs. Progression is usually rapid despite therapy, with a median overall patient survival of only eight months. Thus, compositions and methods for the early and accurate diagnosis and treatment of leukemia, particularly AMKL, are needed.
SUMMARY OF THE INVENTION
Compositions and methods associated with the diagnosis and treatment of leukemia are provided. The invention discloses the identification, cloning and sequencing of human nucleotide sequences corresponding to the t(1;22)(p13;q13) chromosomal translocation event which occurs in individuals with acute megakaryoblastic leukemia (AMKL). The rearrangement recombines sequences from the RNA-binding motifprotein-15 gene (RBM15) on chromosome 1p13 with those from the Megakaryoblastic Leukemia-1 gene (MKL1) on chromosome 22q13. As a result of the t(1;22)(p13;q13) rearrangement, two fusion genes, one on each of the two derivative chromosomes (der(1) and der(22)), are produced. The first fusion gene, designated RBM15-MKL1, resides on der(22) and comprises a 5′ portion of the RBM15 and a 3′ segment of the MKL1. The second fusion gene, designated as MKL1-RBM15, resides on der(1) and comprises a 5′ portion of the MKL1 and a 3′ segment of the RBM15. Both fusion genes are transcribed. A single transcript is expressed from RBM15-MKL1. Two transcripts are found to be expressed from MKL1-RBM15. Isolated nucleotide molecules comprising the nucleotide sequences of the RBM15-MKL1 gene transcript and the two MKL1-RBM15 gene transcripts, MKL1-RBM15S and MKL1-RBM15S+AE are provided. Additionally provided are the amino acid sequences of the fusion proteins encoded by the RBM15-MKL1 transcript and the two MKL1-RBM15 transcripts.
Utilizing the sequences of the present invention, the present invention provides methods of identifying the presence of nucleic acids containing the RBM15-MKL1 fusion gene and/or MKL1-RBM15 fusion gene and the transcripts of these fusion genes in a sample. Such methods can be used in diagnosis and treatment, for example, to determine if particular cells or tissues express RBM15-MKL1 or MKL1-RBM15 coding sequences, or diagnostic assays to determine if a mammal has leukemia or a genetic predisposition to (i.e., is at an increased risk of developing) leukemia.
The RBM15-MKL1 and MKL1-RBM15 fusion proteins and polypeptide sequences of the invention, whether produced by host/vector systems or otherwise, can be used to produce antibodies which specifically recognize (i.e., bind) the RBM15-MKL1 and MKL1-RBM15 fusion proteins, respectively. The invention provides methods of identifying the presence of nucleic acids encoding the RBM15-MKL1 fusion protein and/or MKL1-RBM15 fusion protein in a sample involving the use of such antibodies. Such methods find use in diagnosis and treatment of AMKL, for example, to determine if particular cells or tissues express the RBM15-MKL1 fusion protein and/or the MKL1-RBM15 fusion proteins and to inhibit the activity of these fusion proteins.
The present invention also provides for transgenic cells and animals, preferably mice, which (a) contain and express an RBM15-MKL1 fusion gene derived from an exogenous source and subsequently introduced into the genome of the cell or animal, (b) contain and express a gene encoding an RBM15-MKL1 fusion protein derived from an exogenous source and subsequently introduced into the genome of the cell or animal, (c) contain and express an MKL1-RBM15 fusion gene derived from an exogenous source and subsequently introduced into the genome of the cell or animal (d) contain and express a gene encoding an MKL1-RBM15 fusion protein derived from an exogenous source and subsequently introduced into the genome of the cell or animal, (e) contain and express both a gene encoding an MKL1-RBM15 fusion protein and a gene encoding an RBM15-MKL1 fusion protein, with both genes derived from an exogenous source and subsequently introduced into the genome of the animal, (f) knock-out the expression of the RBM15 and/or MKL1 genes, or (g) knock-out the expression of the RBM15-MKL1 and/or MKL1-RBM15 fusion proteins in an AMKL cell line. Methods of utilizing such cells and mice to identify and test carcinogenic or therapeutic compositions are also described herein.
The nucleotide sequences of the RBM15-MKL1 and MKL1-RBM15 genes and the coding sequences of the RBM15-MKL1 and the two MKL1-RBM15 fusion proteins of the invention can also be utilized to design and prepare agents which specifically inhibit the expression of the RBM15-MKL1 or MKL1-RBM15 genes in cells for therapeutic and other purposes. The RBM15-MKL1 and MKL1-RBM15 nucleotide sequences of the invention can be further utilized in methods of producing the RBM15-MKL1 and MKL1-RBM15 fusion proteins, respectively.
The present invention further provides methods for isolating and identifying the natural ligand(s) and gene targets bound by the RBM15-MKL1 and MKL1-RBM15 fusion proteins, and for identifying derivatives of the ligand(s) or synthetic compounds that act to inhibit the action of the RBM15-MKL1 and MKL1-RBM15 fusion proteins.
Additionally provided are compartmentalized kits to receive in close confinement one or more containers containing the reagents used in one or more of the above described detection methods.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of the normal RBM15 and MKL1 proteins, and the fusion proteins formed by t(1;22). The locations of the fusion junctions in the proteins are indicated by vertical arrows. The 931-aa MKL1 protein (predicted mass, 98.9 kDa; SEQ ID. NO: 14) contains a bipartite nuclear localization signal (N, residues 14-31 of SEQ ID NO: 14), a single SAP DNA-binding motif (residues 347-381 of SEQ ID NO: 14), a coiled-coil region (CC, residues 521-563 of SEQ ID NO: 14), and a long C-terminal proline-rich segment (P, residues 564-811 of SEQ ID NO: 14). The three isofomis of RBM15 (RBM15S, RBM15S+AE and RBM15L) share an identical 2863-bp 5′ coding sequence (nucleotides 84 to 2946 of SEQ ID Nos. 7, 9, and 11) and differ only in their extreme C-termini, distal to the location of the t(1;22) fusion junction, due to splicing of alternative exons. Specifically, RBM15L, the longest RBM15 transcript (approx. 9 kb), contains a unique 416-bp 3′ exon (nucleotides 2947 to 3362 of SEQ ID NO: 11) and encodes a predicted 957-aa protein (mass, 104.6 kDa; SEQ ID NO: 12), whereas the shortest transcript, RBM15S (approx. 4 kb), possesses a divergent 3′ sequence (nucleotides 2947 to 3312 of SEQ ID NO: 7) and encodes a 969-aa protein (mass, 106.2 kDa; SEQ ID NO: 8). Detailed analysis of the approximately 4-kb RBM15 transcript revealed a variant, RBM15S+AE (short transcript plus alternative exon), which contains an additional 111-bp exon (nucleotides 2947 to 3057 of SEQ ID NO: 9) interposed between the 2,863-bp common sequence and the 366-bp sequence in RBM15S and encodes a polypeptide of 977 aa (mass, 107.1 kDa; SEQ ID NO: 10). All RBM15 isoforms contain three RNA recognition motifs (RRM; residues 170-252, 374-451 and 455-529 of SEQ ID Nos. 8, 10, and 12), a bipartite nuclear localization signal (N, residues 716-733 of SEQ ID Nos. 8, 10, and 12), and a SPOC domain (S, residues 714-954 of SEQ ID Nos. 8, 10, and 12). Several regions in RBM15 of potential functional importance are characterized by a high content of specific aa, including a glycine/serine-rich segment (residues 60-166 of SEQ ID Nos. 8, 10, and 12), a small proline-rich (LPPPPPPPLP) motif (residues 315-324 of SEQ ID Nos. 8, 10, and 12), an arginine-rich portion (residues 616-732 of SEQ ID Nos. 8, 10, and 12), and a short C-terminal serine-rich segment (GSSDSRSSSSSAASD) at amino acids 865-879 of SEQ ID Nos. 8, 10, and 12. The 1883-aa RBM15-MKL1 chimeric protein (mass, 203.1 kDa; SEQ ID NO: 2) is comprised of the common N-terminal portion of RBM15 (residues 1 to 954 of SEQ ID Nos. 8, 10, and 12) and all but the first two residues of MKL1, thus containing all identified motifs of each normal protein. By contrast, the reciprocal MKL1-RBM15 fusions contain only the first two aa of MKL1 fused to the short C-terminal sequences of either RBM15S or RBM15S+AE, and are predicted to encode peptides of only 17 (SEQ ID NO: 4) and 25 (SEQ ID NO: 6) amino acids, respectively. The portions of the schematic illustrating the alternative C-termini of RBM15 and the predicted MKL1-RBM15 fusion peptides are enlarged for clarity, and are thus not to scale.



DETAILED DESCRIPTION OF THE INVENTION
Compositions and methods for the identification, diagnosis, and treatment of leukemia or a genetic predisposition to leukemia are provided. The present invention is based on the discovery of two reciprocal fusion genes, RBM15-MKL1 and MKL1-RBM15, that result from the t(1;22)(p13;q13) chromosomal translocation event associated with acute megakaryoblastic leukemia (AMKL). In particular, the invention provides the novel nucleotide sequences of a transcript of the RBM15-MKL1 fusion gene (SEQ ID NO: 1) and two transcripts, MKL1-RBM15S (SEQ ID NO: 3) and MKL1-RBM15S+AE (SEQ ID NO: 5), of the MKL1-RBM15 fusion gene. Additionally provided are the amino acid sequences (SEQ ID NOS: 2, 4, and 6, respectively) of the fusion proteins encoded by such nucleotide sequences. Such nucleotide sequences and amino acid sequences find use, for example, in methods for detecting the t(1;22)(p13;q13) chromosomal translocation event associated with AMKL, methods for identifying agents that bind to the fusion proteins and methods for identifying agents useful for treating AMKL.
In addition to the RBM15-MKL1 and MKL1-RBM15 nucleotide and amino acid sequences, the invention provides isolated nucleotide molecules comprising nucleotide sequences of three transcripts of the RBM15 gene, RBM15S (SEQ ID NO: 7), RBM15S+AE (SEQ ID NO: 9), and RBM15L (SEQ ID NO: 11) and the nucleotide sequence of MKL1 (SEQ ID NO: 13). Further provided are isolated proteins comprising the amino acids sequences of the proteins encoded thereby, RBM15S (SEQ ID NO: 8), RBM15S+AE (SEQ ID NO: 10), and RBM15L (SEQ ID NO: 12) and MKL1 (SEQ ID NO: 14) respectively. Such nucleotide and amino acid sequences find use, for example, in methods for detecting the t(1;22)(p13;q13) chromosomal translocation event associated with AMKL and methods for identifying agents useful for treating AMKL.
The RBM15 and MKL1 proteins, as well as the noted fusion proteins derived from RBM15 and MKL1, are contemplated to act as transcription factors which bind to corresponding DNA regulatory sequences. Thus these proteins may be used to regulate the expression of genes that include the regulatory DNA sequences that these proteins recognize. Methods for identifying such regulatory DNA sequences based on their ability to bind RBM15, MKL1, RBM15-MKL1 and MKL1-RBM15 are also included in the present invention.
The nucleotide and amino acid sequences of the invention are set forth in the sequence listing. Below is a brief description of the sequences in the sequences listing.

    
    
        SEQ ID NO: 1 is the nucleotide sequence RBM15-MKL1 cDNA. The open reading frame is from nucleotide 84 through nucleotide 5732.
        SEQ ID NO: 2 is the amino acid sequence of the RBM15-MKL1 fusion protein.
        SEQ ID NO: 3 is the nucleotide sequence MKL1-RBM15S cDNA. The open reading frame is from nucleotide 551 through nucleotide 601.
        SEQ ID NO: 4 is the amino acid sequence of the MKL1 -RBM15S fusion protein.
        SEQ ID NO: 5 is the nucleotide sequence MKL1-RBM15S+AE cDNA. The open reading frame is from nucleotide 551 through nucleotide 625.
        SEQ ID NO: 6 is the amino acid sequence of the MKL1-RBM15S+AE fusion protein.
        SEQ ID NO: 7 is the nucleotide sequence RBM15S cDNA. The open reading frame is from nucleotide 84 through nucleotide 2990.
        SEQ ID NO: 8 is the amino acid sequence of the RBM15S protein.
        SEQ ID NO: 9 is the nucleotide sequence RBM15S+AE cDNA. The open reading frame is from nucleotide 84 through nucleotide 3014.
        SEQ ID NO: 10 is the amino acid sequence of the RBM15S+AE protein.
        SEQ ID NO: 11 is the nucleotide sequence RBM15L cDNA. The open reading frame is from nucleotide 84 through nucleotide 2954.
        SEQ ID NO: 12 is the amino acid sequence of the RBM15L protein.
        SEQ ID NO: 13 is the nucleotide sequence MKL1 cDNA. The open reading frame is from nucleotide 551 through nucleotide 3346.
        SEQ ID NO: 14 is the amino acid sequence of the MKL1 protein.
        SEQ ID NO: 15 is the nucleotide sequence of the synthetic oligonucleotide primer herein designated MKL1-294R.
        SEQ ID NO: 16 is the nucleotide sequence of the synthetic oligonucleotide primer herein designated MKL1-73R.
        SEQ ID NO: 17 is the nucleotide sequence of the synthetic oligonucleotide primer herein designated MKL1-59R.
        SEQ ID NO: 18 is the nucleotide sequence of the synthetic oligonucleotide primer herein designated RBM15(S)-2746F.
        SEQ ID NO: 19 is the nucleotide sequence of the synthetic oligonucleotide primer herein designated MKL1-204R.
        SEQ ID NO: 20 is the nucleotide sequence of the synthetic oligonucleotide primer herein designated MKL1-F.
        SEQ ID NO: 21 is the nucleotide sequence of the synthetic oligonucleotide primer herein designated RBM15(S)-2930R.
        SEQ ID NO: 22 is the nucleotide sequence of the synthetic oligonucleotide primer herein designated RBM15(L)-1636R.
        SEQ ID NO: 23 is the nucleotide sequence of the synthetic oligonucleotide primer herein designated RBM15-1118F.
        SEQ ID NO: 24 is the nucleotide sequence of the synthetic oligonucleotide primer herein designated RBM15-1551R.
        SEQ ID NO: 25 is the nucleotide sequence of the synthetic oligonucleotide primer herein designated RBM15-2831F.
        SEQ ID NO: 26 is the nucleotide sequence of the synthetic oligonucleotide primer herein designated RBM15-3149R.
        SEQ ID NO: 27 is the nucleotide sequence of the synthetic oligonucleotide primer herein designated RBM15-1616F.
        SEQ ID NO: 28 is the nucleotide sequence of the synthetic oligonucleotide primer herein designated RBM15-2004R.
        SEQ ID NO: 29 is the nucleotide sequence of the synthetic oligonucleotide primer herein designated MKL1-155F.
    
    


Based on these observations, one embodiment of the present invention provides a first isolated nucleotide molecule comprising the coding sequence (SEQ ID NO: 1) of the RBM15-MKL1 fusion protein, which encodes the RBM15-MKL1 fusion protein (SEQ ID NO: 2), a second isolated nucleotide molecule, comprising the coding sequence (SEQ ID NO: 3) of the MKL1-RBM15S fusion protein, which encodes the MKL1-RBM15S fusion protein (SEQ ID NO: 4), a third isolated nucleotide molecule comprising the coding sequence (SEQ ID NO: 5) of the MKL1-RBM15S+AE fusion protein which encodes the MKL1-RBM15S+AE fusion protein (SEQ ID NO: 6), a fourth isolated nucleotide molecule, comprising the coding sequence (SEQ ID NO: 7) of the RBM15S protein, which encodes the RBM15S protein (SEQ ID NO: 8), a fifth isolated nucleotide molecule comprising the coding sequence (SEQ ID NO: 9) of the RBM15S+AE protein which encodes the RBM15S+AE protein (SEQ ID NO: 10), a sixth isolated nucleotide molecule, comprising the coding sequence (SEQ ID NO: 11) of the RBM15L protein, which encodes the RBM15L protein (SEQ ID NO: 12), and a seventh isolated nucleotide molecule comprising the coding sequence (SEQ ID NO: 13) of the MKL1 protein, which encodes the MKL1 protein (SEQ ID NO: 14).
It is recognized that nucleotide molecules and proteins of the invention will have a nucleotide or an amino acid sequence sufficiently identical to a nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, or 13 or to an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14. The term “sufficiently identical” is used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of identical or equivalent (e.g., with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain and/or common functional activity. For example, amino acid or nucleotide sequences that contain a common structural domain having at least about 45%, 55%, or 65% identity, preferably 75% identity, more preferably 85%, 95%, or 98% identity are defined herein as sufficiently identical.
To determine the percent identity of two amino acid sequences or of two nucleic acids, the sequences are aligned for optimal comparison purposes. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity=number of identical positions/total number of positions (e.g., overlapping positions)×100). In one embodiment, the two sequences are the same length. The percent identity between two sequences can be determined using techniques similar to those described below, with or without allowing gaps. In calculating percent identity, typically exact matches are counted.
The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A preferred, nonlimiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul el al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
The invention encompasses RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, and MKL1 nucleic acid molecules and fragments thereof. Nucleic acid molecules that are fragments of these nucleotide sequences are also encompassed by the present invention. By “fragment” is intended a portion of the nucleotide sequence encoding an RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L or MKL1 protein. A fragment of an RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L or MKL1 nucleotide sequence may encode a biologically active portion of an RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L or MKL1 protein, or it may be a fragment that can be used as a hybridization probe or PCR primer using methods disclosed below. A biologically active portion of an RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L or MKL1 protein can be prepared by isolating a portion of one of the RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, and MKL1 nucleotide sequences of the invention, expressing the encoded portion of the RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L or MKL1 protein (e.g., by recombinant expression in vitro), and assessing the activity of the encoded portion of the RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, and MKL1 protein. Nucleic acid molecules that are fragments of an RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, and MKL1 nucleotide sequence comprise at least about 15, 20, 50, 75, 100, 200, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4500, 5000, 5500, 6000, or 6500 nucleotides, or up to the number of nucleotides present in a full-length nucleotide sequence disclosed herein (for example, 6836, 923, 1034, 3312, 3423, 3383, and 4447 nucleotides for SEQ ID NOS: 1, 3, 5, 7, 9, 11, and 13, respectively) depending upon the intended use.
It is understood that isolated fragments include any contiguous sequence not disclosed prior to the invention as well as sequences that are substantially the same and which are not disclosed. Accordingly, if an isolated fragment is disclosed prior to the present invention, that fragment is not intended to be encompassed by the invention. When a sequence is not disclosed prior to the present invention, an isolated nucleic acid fragment is at least about 12, 15, 20, 25, or 30 contiguous nucleotides. Other regions of the nucleotide sequence may comprise fragments of various sizes, depending upon potential homology with previously disclosed sequences.
A fragment of an RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L or MKL1 nucleotide sequence that encodes a biologically active portion of an RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, or MKL1 protein of the invention will encode at least about 15, 25, 30, 50, 75, 100, 125, 150, 175, 200, 250, or 300 contiguous amino acids, or up to the total number of amino acids present in a full-length RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, or MKL1 protein of the invention (for example, 1883, 17, 25, 969, 977, 957, and 931 amino acids for SEQ ID NOS: 2, 4, 6, 8, 10, 12, and 14, respectively). Fragments of an RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L and MKL1 nucleotide sequence that are useful as hybridization probes for PCR primers generally need not encode a biologically active portion of an RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, and MKL1 protein, respectively.
Nucleic acid molecules that are variants of the nucleotide sequences disclosed herein are also encompassed by the present invention. “Variants” of the RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, and MKL1 nucleotide sequences include those sequences that encode the RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, and MKL1 proteins, respectively, disclosed herein but that differ conservatively because of the degeneracy of the genetic code. These naturally occurring allelic variants can be identified with the use of well-known molecular biology techniques, such as polymerase chain reaction (PCR) and hybridization techniques as outlined below. Variant nucleotide sequences also include synthetically derived nucleotide sequences that have been generated, for example, by using site-directed mutagenesis but which still encode the RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, or MKL1 protein disclosed in the present invention as discussed below. Generally, nucleotide sequence variants of the invention will have at least about 45%, 55%, 65%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97, 98%, or 99% identity to a particular nucleotide sequence disclosed herein. A variant RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L or MKL1 nucleotide sequence will encode a variant RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, or MKL1 protein, respectively, that has an amino acid sequence having at least about 45%, 55%, 65%, 75%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97, 98%, or 99% identity to the amino acid sequence of an RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, or MKL1 protein disclosed herein.
In addition to the RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L and MKL1 nucleotide sequences shown in SEQ ID NOS: 1, 3, 5, 7, 9, 11, and 13 , it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of RBM5-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, or MKL1 proteins may exist within a population (e.g., the human population). Such genetic polymorphism in an RBM15-MKL1, MKL1-RBM15, RBM15, and MKL1 gene may exist among individuals within a population due to natural allelic variation. An allele is one of a group of genes that occur alternatively at a given genetic locus. As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding an RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, or MKL1 protein, preferably a mammalian RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, or MKL1 protein. As used herein, the phrase “allelic variant” refers to a nucleotide sequence that occurs at an RBM15-MKL1, MKL1-RBM15, RBM15, and MKL1 locus or to a polypeptide encoded by the nucleotide sequence. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of the RBM15-MKL1, MKL1-RBM15, RBM15, and MKL1 gene. Any and all such nucleotide variations and resulting amino acid polymorphisms or variations in an RBM15-MKL1, MKL1-RBM15, RBM15, and MKL1 amino acid sequence that are the result of natural allelic variation and that do not alter the functional activity of RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, and MKL1 proteins are intended to be within the scope of the invention.
Moreover, nucleic acid molecules encoding RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, and MKL1 proteins from other species (RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, and MKL1 homologues), which have a nucleotide sequence differing from that of the RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, and MKL1 sequences disclosed herein, are intended to be within the scope of the invention. For example, nucleic acid molecules corresponding to natural allelic variants and homologues of the human RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L and MKL1 cDNAs of the invention can be isolated based on their identity to the human RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, and MKL1 nucleic acids disclosed herein using the human cDNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions as disclosed herein.
In addition to naturally occurring allelic variants of the RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, and MKL1 sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of the invention thereby leading to changes in the amino acid sequence of the encoded RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, and MKL1 proteins, respectively, without altering the biological activity of the RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, and MKL1 proteins. Thus, an isolated nucleic acid molecule encoding an RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, or MKL1 protein having a sequence that differs from that of SEQ ID NOS: 2, 4, 6, 8, 10, 12, or 14, respectively, can be created by introducing one or more nucleotide substitutions, additions, or deletions into the corresponding nucleotide sequence disclosed herein, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. Such variant nucleotide sequences are also encompassed by the present invention.
For example, preferably, conservative amino acid substitutions may be made at one or more predicted, preferably nonessential amino acid residues. A “nonessential” amino acid residue is a residue that can be altered from the wild-type sequence of an RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, or MKL1 protein (e.g., the sequence of SEQ ID NOS: 2, 4, 6, 8, 10, 12, or 14, respectively) without altering the biological activity, whereas an “essential” amino acid residue is required for biological activity. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Such substitutions would not be made for conserved amino acid residues, or for amino acid residues residing within a conserved motif.
Alternatively, variant RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, and MKL1 nucleotide sequences can be made by introducing mutations randomly along all or part of an RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L or MKL1 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, or MKL1 biological activity to identify mutants that retain activity. Following mutagenesis, the encoded protein can be expressed recombinantly, and the activity of the protein can be determined using standard assay techniques.
Thus, the nucleotide sequences of the invention include the sequences disclosed herein as well as fragments and variants thereof. The RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, and MKL1 nucleotide sequences of the invention, and fragments and variants thereof, can be used as probes and/or primers to identify and/or clone RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, and MKL1 homologues in other cell types, e.g., from other tissues, as well as homologues from other mammals. Such probes can be used to detect transcripts or genomic sequences encoding the same or identical proteins.
In this manner, methods such as PCR, hybridization, and the like can be used to identify such sequences having substantial identity to the sequences of the invention. See, for example, Sambrook et al. (1989) Molecular Cloning: Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.) and Innis, et al. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, NY). RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, and MKL1 nucleotide sequences isolated based on their sequence identity to the RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L and MKL1 nucleotide sequences set forth herein or to fragments and variants thereof are encompassed by the present invention.
In a hybridization method, all or part of a known RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L or MKL1 nucleotide sequence can be used to screen cDNA or genomic libraries. Methods for construction of such cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). The so-called hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32P, or any other detectable marker, such as other radioisotopes, a fluorescent compound, an enzyme, or an enzyme co-factor. Probes for hybridization can be made by labeling synthetic oligonucleotides based on the known RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, and MKL1 nucleotide sequence disclosed herein. Degenerate primers designed on the basis of conserved nucleotides or amino acid residues in a known RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L and MKL1 nucleotide sequence or encoded amino acid sequence can additionally be used. The probe typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, or 400 consecutive nucleotides of an RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, and MKL1 nucleotide sequence of the invention or a fragment or variant thereof. Preparation of probes for hybridization is generally known in the art and is disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.), herein incorporated by reference.
As used herein, the term “hybridizes under stringent conditions” is intended to describe conditions for hybridization and washing under which nucleotide sequences having at least about 60%, 65%, 70%, preferably 75% identity to each other typically remain hybridized to each other. Such stringent conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology (John Wiley & Sons, New York (1989)), 6.3.1-6.3.6. A preferred, non-limiting example of stringent hybridization conditions is hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 50-65° C. In another preferred embodiment, stringent conditions comprise hybridization in 6×SSC at 42° C., followed by washing with 1×SSC at 55° C. Preferably, an isolated nucleic acid molecule that hybridizes under stringent conditions to an RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, or MKL1 nucleotide sequence of the invention corresponds to a naturally occurring nucleic acid molecule. As used herein, a “naturally occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
Thus, in addition to the RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, and MKL1 nucleotide sequences disclosed herein and fragments and variants thereof, the isolated nucleic acid molecules of the invention also encompass homologous DNA sequences identified and isolated from other cells and/or organisms by hybridization with entire or partial sequences obtained from the RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, and MKL1. By inserting any of the nucleotide sequences of the present invention into an appropriate vector, one skilled in the art can readily produce large quantities of the specific sequence. Alternatively, the RBM15-MKL1, MKL1-RBM15S and MKL1-RBM15S+AE, nucleotide sequences of the invention can be further utilized in methods of producing the RBM15-MKL1, MKL1-RBM15S and MKL1-RBM15S+AE fusion proteins, respectively, by introduction of the appropriate coding sequence into a host/vector expression system. There are numerous host/vectors systems available for the propagation of nucleotide sequences and/or the production of expressed proteins. These include, but are not limited to, plasmid and viral vectors, and prokaryotic and eukaryotic host. One skilled in the art can readily adapt any host/vector system which is capable of propagating or expressing heterologous DNA to produce or express the sequences of the present invention. Of course, the RBM15-MKL1, MKL1-RBM15S and MKL1-RBM15S+AE fusion proteins or polypeptides derived therefrom may also be produced by other means known in the art such as, for example, chemical synthesis or in vitro transcription/translation.
Also provided by the present invention are an isolated RBM15-MKL1 fusion protein (SEQ ID NO: 2), an isolated MKL1-RBM15S fusion protein (SEQ ID NO: 4), and an isolated MKL1-RBM15S+AE fusion protein (SEQ ID NO: 6), an isolated RBM15S protein (SEQ ID NO: 8), an isolated RBM15S+AE protein (SEQ ID NO: 10), an isolated RBM15L protein (SEQ ID NO: 12), and an isolated MKL1 protein (SEQ ID NO: 14), which are encoded by their cognate nucleotides, that is, by SEQ ID NO: 1, 3, 5, 7, 9, 11, and 13, respectively. Synthetic oligopeptides derived from SEQ ID NO: 2, 4, 6, 8, 10, 12, and 14 are also provided in this embodiment of the invention.
An “isolated” or “purified” nucleic acid molecule or protein, or biologically active portion thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. Preferably, an “isolated” nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For purposes of the invention, “isolated” when used to refer to nucleic acid molecules excludes isolated chromosomes. For example, in various embodiments, the isolated nucleic acid molecules can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
An isolated protein that is substantially free of cellular material includes preparations of RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, or MKL1 protein having less than about 30%, 20%, 10%, or 5% (by dry weight) of non-like protein (also referred to herein as a “contaminating protein”). When the RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, or MKL1 protein or biologically active portion thereof is recombinantly produced, preferably, culture medium represents less than about 30%, 20%, 10%, or 5% of the volume of the protein preparation. When RBM15-MKL1, MKL1-RBM15S, MKL1-RBM15S+AE, RBM15S, RBM15S+AE, RBM15L, or MKL1 protein is produced by chemical synthesis, preferably the protein preparations have less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-like chemicals.
In Example 1, the present invention provides evidence that the nucleotide sequences containing the RBM15-MKL1 and MKL1-RBM15 fusion genes are present in patients with t(1;22) AMKL. Based on this observation, the present invention provides methods of assaying for the presence of nucleotide sequences containing the RBM15-MKL1 and MKL1-RBM15 fusions in a sample and thus provides an assay for the detection of t(1;22) leukemias, as explained in Example 1. The methods of the invention can involve any means known in the art for detecting the presence of specific nucleotide sequences in a sample including, but not limited to, nucleic acid hybridization and detection methods (e.g., “Southerns,” “Northerns” and the like) fluorescence in situ hybridization (FISH) and detection methods, or polymerase chain reaction (PCR) amplification and detection methods, particularly reverse transcriptase-polymerase chain reaction amplification (RT-PCR).
One example of the assay methods of the present invention which are used to detect RBM15-MKL1 or MKL1-RBM15 fusion gene are based on the preferential amplification of sequences within a sample which contain the nucleotide sequences encoding the RBM15-MKL1, MKL1-RBM15S or MKL1-RBM15S+AE fusion proteins. In one embodiment of the invention, RT-PCR is utilized to detect the t(1,22) rearrangement that is associated with AMLK. The method involves the use of RT-PCR to detect the presence of transcripts from the RBM15-MKL1 or MKL1-RBM15 fusion genes. The method involves reverse transcription via reverse transcriptase of an RNA sample from a patient to produce cDNA. For reverse transcription, an oligo-dT primer can be use, or alternatively, a primer designed to specifically anneal to RBM15-MKL1 mRNA, MKL1-RBM15SmRNA, or MKL1-RBM15S+AEmRNA can be employed to prime cDNA synthesis. Such primers can be designed from the nucleotide sequences of the invention as set forth in SEQ ID NOS: 1, 3, and 5 using methods known to those of ordinary skill in the art. Then, PCR amplification of the cDNA can be performed utilizing primers designed to amplify at least a portion of the nucleotide sequences of to RBM15-MKL1, MKL1-RBM15S or MKL1-RBM15S+AE. The amplified cDNA can be detected by methods known in the art such as, for example, agarose gel electrophoresis and ethidium bromide staining. The detection of the desired cDNA corresponding to at least a portion of the RBM15-MKL1, MKL1-RBM15S or MKL1-RBM15S+AE indicates that the sample is from a patient with the t(1,22) rearrangement.
The methods of the invention involve the use of PCR amplification, particularly RT-PCR. Methods for PCR amplification are known in the art. Oligonucleotide primers can be designed for use in PCR reactions to amplify corresponding DNA sequences from genomic DNA or cDNA extracted from any organism of interest. Methods for PCR amplification and for designing PCR primers are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See also Innis et al., eds. (1990) PCR Protocols: A Guide to Methods and Applications (Academic Press, New York); Innis and Gelfand, eds. (1995) PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York). Other known methods of PCR that can be used in the methods of the invention include, but are not limited to, methods using paired primers, nested primers, single specific primers, degenerate primers, gene-specific primers, mixed DNA/RNA primers, vector-specific primers, partially mismatched primers, and the like.
In addition to methods which rely on the amplification of a target sequence, the present invention further provides methods for identifying nucleic acids containing the RBM15-MKL1 fusion gene which do not require sequence amplification and are based on the known methods of Southern (DNA:DNA) and Northern (DNA:RNA) blot hybridizations, and FISH of chromosomal material, using probes derived from the nucleotide sequences of the invention. Additionally, other nucleotide sequences of chromosomes 1 and 22 that are known to art and for which are disclosed herein to occur in the vicinity of the chromosomal breakpoint for the t(1;22)(p13;q13) chromosomal translocation event associated with AMKL can be used in the methods of the invention. That is, nucleic acid probes can be used that comprise nucleotide sequences in proximity to the t(1;22)(p13;q13) chromosomal translocation event, or breakpoint. By “in proximity to” is intended within about 10 kilobases (kb) of the t(1;22) breakpoint. Such other nucleotide sequences include, but are not limited to, RP11-260A24 (Accession no. AC025987), RP5-1042K10 (Accession no. AL022238), RP11-313L7, RP5-1125M8 (Accession no. AL356387), RP4-665N5, RP4-743K1, RP11-50F6, RP3-377F16 (Accession no. Z93783), RP4-591N18 (Accession no. AL031594), RP1-229A8 (Accession no. Z86090), and RP4-735G18 (Accession no. AL096703). The clones not identified with Accession numbers are also available from the Roswell Park Cancer Institute (RPCI-BAC library).
In another embodiment of the invention, methods are provided detecting the t(1,22) rearrangement involving FISH (fluorescence in situ hybridization ) of human chromosomal material. For example, a probe that is comprised of nucleotide sequences that span the breakpoint in either a wild-type chromosome 1 or 22 can be used. Such a probe can hybridize to both derivative chromosomes in the case of a t(1,22) rearrangement. Alternatively, two probes, each labeled with a different detection reagent, can be utilized. The first probe is capable of hybridizing to sequences within the region of chromosomal band 1p13, and the second probe is capable of binding to the region of chromosomal band 22q13. The two probes are also selected such that, in a t(1,22) rearrangement, both probes hybridize to the same derivative chromosome, whether it be chromosome 1 or 22. In such a case, a signal from each of the probes is observed on the same chromosome.
The nucleic acid probes of the present invention include DNA as well as RNA probes, such probes being generated using techniques known in the art (Sambrook et al., eds., Molecular Cloning, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1989)). A skilled artisan can employ such known techniques using the RBM15S, RBM15S+AE, RBM15L, MKL1, RBM15-MKL1, MKL1-RBM15S, and MKL1-RBM15S+AE nucleotide sequences herein described, or fragments thereof, as probes.
For nucleic acid probes, examples of detection reagents include, but are not limited to radiolabeled probes, enzymatic labeled probes (horse radish peroxidase, alkaline phosphatase), and affinity labeled probes (biotin, avidin, or steptavidin). For antibodies, examples of detection reagents include, but are not limited to, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the chromophoric, enzymatic, or antibody binding reagents which are capable of reacting with the labeled antibody. One skilled in the art will readily recognize that the antibodies and nucleic acid probes described in the present invention can readily be incorporated into one of the established kit formats which are well known in the art.
The samples used in the detection methods of the present invention include, but are not limited to, cells or tissues, protein, membrane, or nucleic acid extracts of the cells or tissues, and biological fluids such as blood, serum, and plasma. The sample used in the methods of the invention will vary based on the assay format, nature of the detection method, and the tissues, cells or extracts which are used as the sample. Methods for preparing protein extracts, membrane extracts or nucleic acid extracts of cells are well known in the art and can be readily be adapted in order to obtain a sample which is compatible with the method utilized (see, for example, K. Budelier et al., Chapter 2, “Preparation and Analysis of DNA,” M. E. Greenberg et al., Chapter 4, “Preparation and Analysis of RNA” and M. Moos et al., Chapter 10, “Analysis of Proteins,” in Ausubel et al., Current Protocols in Molecular Biology, Wiley Press, Boston, Mass. (1993)). One preferred type of sample which can be utilized in the present invention is derived from isolated lymphoma cells. Such cells can be used to prepare a suitable extract or can be used in procedures based on in situ analysis.
The present invention further provides antibodies specific to epitopes of the RBM15-MKL1, MKL1-RBM15S, or MKL1-RBM15S+AE fusion proteins and methods of detecting the RBM15-MKL1, MKL1-RBM15S, or MKL1-RBM15S+AE fusion proteins, or any combination thereof, that rely on the ability of these antibodies to selectively bind to specific portions of the RBM15-MKL1, MKL1-RBM15S, or MKL1-RBM15S+AE proteins that are unique to that fusion protein. Such antibodies do not bind preferentially to the RBM15 or MKL1 proteins.
The present invention further provides methods of detecting the presence of at least one of the RBM15-MKL1, MKL1-RBM15S, and MKL1-RBM15S+AE fusion proteins. Antibodies can be prepared which recognize a fusion protein of the invention. Such antibodies can be used to detect the presence of the fusion protein in samples from human cells. The methods of the invention involve the use of antibodies that bind to at least one of the fusion proteins of the invention and antibody detection systems that are known to those of ordinary skill in the art. Such methods find use in diagnosis and treatment of AMKL, for example, to determine if particular cells or tissues express the RBM15-MKL1, MKL1-RBM15S, and/or the MKL1-RBM15S+AE fusion proteins.
Conditions for incubating an antibody with a test sample vary depending on the format employed for the assay, the detection methods employed, the nature of the test sample, and the type and nature of the antibody used in the assay. One skilled in the art will recognize that any one of the commonly available immunological assay formats (such as radioimmunoassays, enzyme-linked immunosorbent assays, diffusion based ouchterlony, or rocket inmunofluorescent assays) can readily be adapted to employ the antibodies of the present invention. Examples of such assays can be found in Chard, T., An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science Publishers, Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques in Immunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).
In another embodiment of the immunoassays of the invention, the anti-RBM15-MKL1 antibody, the anti-MKL1-RBM15S antibody, or the anti-MKL1-RBM15S+AE antibody is immobilized on a solid support. Examples of such solid supports include, but are not limited to, plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, and acrylic resins, such as polyacrylamide and latex beads. Techniques for coupling antibodies to such solid supports are well known in the art (see, for example, Weir, D. M. et al., Handbook of Experimental Immunology, 4th Ed., Blackwell Scientific Publications, Oxford, England, Chapter 10 (1986)).
Additionally, one or more of the antibodies used in the above described methods can be detectably labeled prior to use. Antibodies can be detectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, etc.), enzymatic labels (such as horse radish peroxidase, alkaline phosphatase, etc.) fluorescent labels (such as FITC or rhodamine, etc.), paramagnetic atoms, etc. Procedures for accomplishing such labeling are well-known in the art; see, for example, Stemberger, L. A. et al., J. Histochem. Cytochem. 18:315-333 (1970); Bayer, E. A. et al., Meth. Enzym. 62:308-315 (1979); Engrall, E. et al., J. Immunol. 109:129-135 (1972); Goding, J. W., J. Immunol. Meth. 13:215-226 (1976).
The present invention further includes methods for selectively killing cells expressing the RBM15-MKL1 fusion protein, the MKL1-RBM15S fusion protein, and/or the MKL1-RBM15S+AE fusion protein by, for example, contacting a cell expressing the RBM15-MKL1, MKL1-RBM15S, and/or MKL1-RBM15S+AE fusion protein with a toxin derivatized antibody, wherein the antibody is capable of selectively binding to the fusion protein with only weak or no binding to non-fusion RBM15 or MKL1 protein. An example of such an antibody is toxin derivatized antibodies which bind to the RBM15-MKL1 fusion protein junction. As used herein, an antibody is said to be “toxin-derivatized” when the antibody is covalently attached to a toxin moiety. Procedures for coupling such moieties to a molecule are well known in the art. The binding of a toxin derivatized antibody to a cell brings the toxin moiety into close proximity to the cell and thereby promotes cell death. By providing such an antibody molecule to a mammal, the cell expressing the fusion protein can be preferentially killed. Any suitable toxin moiety may be employed; however, it is preferable to employ toxins such as, for example, the ricin toxin, the cholera toxin, the diphtheria toxin, radioisotopic toxins, or membrane-channel-forming toxins.
The antibodies or toxin-derivatized antibodies of the present invention may be administered to a mammal intravenously, intramuscularly, subcutaneously, enterally, topically or parenterally. When administering antibodies or peptides by injection, the administration may be by continuous injections, or by single or multiple injections.
The antibodies or toxin-derivatized antibodies of the present invention are intended to be provided to recipient mammal in a “pharmaceutically acceptable form” in an amount sufficient to “therapeutically effective.” An amount is said to be therapeutically effective if the dosage, route of administration, etc. of the agent are sufficient to preferentially kill a portion of the cells expressing the RBM15-MKL1 or MKL1-RBM15 fusion protein. An antibody is said to be in a “pharmacologically acceptable form” if its administration can be tolerated by a recipient patient. The antibodies of the present invention can be formulated according to known methods of preparing pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington's Pharmaceutical Sciences, 16th ed., Osol, A., ed., Mack, Easton Pa. (1980). In order to form a pharmaceutically acceptable composition which is suitable for effective administration, such compositions will contain an effective amount of an antibody of the present invention together with a suitable amount of carrier. In addition to carriers, the antibodies of the present invention may be supplied in humanized form. Humanized antibodies may be produced, for example by replacing an immunogenic portion of an antibody with a corresponding, but non-immunogenic portion (i.e., chimeric antibodies) (Robinson, R. R. et al., International Patent Publication PCT/US86/02269; Akira, K. et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison, S. L. et al., European Patent Application 173,494; Neuberger, M. S. et al., PCT Application WO 86/01533; Cabilly, S. et al., European Patent Application 125,023; Better, M. et al., Science 240:1041-1043 (1988); Liu, A. Y. et al., Proc. Natl. Acad. Sci. USA 84:3439-3443 (1987); Liu, A. Y. et al., J. Immunol. 139:3521-3526 (1987); Sun, L. K. et al., Proc. Natl. Acad. Sci. USA 84:214-218 (1987); Nishimura, Y. et al., Cancer Res. 47:999-1005 (1987); Wood, C. R. et al., Nature 314:446-449 (1985)); Shaw el al., J. Natl. Cancer Inst. 80:1553-1559 (1988).
In providing a patient with an antibody or toxin-derivatized antibody, the dosage of administered agent will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition, previous medical history, etc. In general, it is desirable to provide the recipient with a dosage of the antibody which is in the range of from about 1 pg/kg to 10 mg/kg (body weight of patient), although a lower or higher dosage may be administered.
The present invention also encompasses antisense nucleic acid molecules, i.e., molecules that are complementary to a sense nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule, or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid. The antisense nucleic acid can be complementary to an entire coding strand, or to only a portion thereof, e.g., all or part of the protein coding region (or open reading frame). An antisense nucleic acid molecule can be antisense to a noncoding region of the coding strand of a nucleotide sequence encoding a protein of interest. The noncoding regions are the 5′ and 3′ sequences that flank the coding region and are not translated into amino acids.
Given the coding-strand sequence encoding, for example, an RBM15-MKL1 fusion protein disclosed herein (e.g., SEQ ID NO: 1), antisense nucleic acids of the invention can be designed according to the rules of Watson and Crick base pairing. The antisense nucleic acid molecule can be complementary to the entire coding region of RBM15-MKL1 mRNA, but more preferably is an oligonucleotide that is antisense to only a portion of the coding or noncoding region of RBM15-MKL1 mRNA. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of RBM15-MKL1 mRNA. A preferred antisense oligonucleotide for selective hybridisation to fusion transcripts will include the region spanning the RBM15 portion of the fusion transcript and the MKL1 portion of the fusion transcript. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleotides in length. An antisense nucleic acid of the invention can be constructed using chemical synthesis and enzymatic ligation procedures known in the art. Similarly, antisense nucleotide molecules can be prepared for the nucleotide sequences encoding the MKL1-RBM15S, and MKL1-RBM15S+AE fusion proteins (SEQ ID NOS: 3 and 5, respectively).
For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, including, but not limited to, for example e.g., phosphorothioate derivatives and acridine substituted nucleotides. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
When used therapeutically, the antisense nucleic acid molecules of the invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a protein of the invention to thereby inhibit expression of the protein, e.g., by inhibiting transcription and/or translation. An example of a route of administration of antisense nucleic acid molecules of the invention includes direct injection at a tissue site. Alternatively, antisense nucleic acid molecules can be modified to target selected cells and then administered systemically. For example, antisense molecules can be linked to peptides or antibodies to form a complex that specifically binds to receptors or antigens expressed on a selected cell surface. The antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations of the antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
An antisense nucleic acid molecule of the invention can be an α-anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual β-units, the strands run parallel to each other (Gaultier et al. (1987) Nucleic Acids Res. 15:6625-6641). The antisense nucleic acid molecule can also comprise a 2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res. 15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBS Lett. 215:327-330).
In another embodiment of the present invention, methods are provided for modulating the translation of at least one RNA selected from the group consisting of those RNAs encoding the RBM15-MKL1, MKL1-RBM15S, or MKL1-RBM15S+AE fusion protein in the cell. Specifically, such methods comprise introducing into a cell a DNA sequence which is capable of transcribing RNA which is complimentary to the mRNA encoding either the RBM15-MKL1, MKL1-RBM15S, or MKL1-RBM15S+AE fusion protein. By introducing such a sequence into a cell, antisense RNA will be produced that will hybridize to RBM15-MKL1, MKL1-RBM15S, or MKL1-RBM15S+AE mRNA and block the translation of the RBM15-MKL1 or MKL1-RBM15 fusion protein, respectively. Antisense cloning has been described elsewhere in more detail by Methis et al., Blood 82:1395-1401 (1993); Stein et al., Science 261:1004-1012 (1993); Mirabella et al., Anti-Cancer Drug Design 6:647-661 (1991); Rosenberg et al., Nature 313:703-706 (1985); Preiss et al., Nature 313:27-32 (1985), Melton, Proc. Natl. Acad. Sci. USA 82:144-148 (1985) and Kim et al., Cell 42:129-138 (1985). Transcription of the introduced DNA will result in multiple copies of the antisense RNA being generated. By controlling the level of transcription of antisense RNA, and the tissue specificity of expression via promoter selection or gene targeting of the antisense expression sequence, one skilled in the art can regulate the level of translation of the RBM15-MKL1, MKL1-RBM15S, and/or MKL1-RBM15S+AE fusion proteins in specific cells within a patient. In a related method, one or more synthetic antisense oligonucleotides that are complementary to the RBM15-MKL1, MKL1-RBM15S, and/or MKL1-RBM15S+AE coding sequences of the invention, optionally including chemical modifications designed to stabilize the oligonucleotide or enhance its uptake into cells, are administered to cells of a patient by known methods (see, for example, R. W. Wagner, Nature 372:333-335 (1994); J. Lisziewicz et al., Proc. Natl. Acad. Sci. (USA) 90:3860-3864 (1993); S. Fitzpatrick-McElligott, Bio/Technology 10: 1036-1040 (1992); E. Uhlmann et al., Chemical Reviews 90:543-583 (1990); and B. Tseng et al., Cancer Gene Therapy 1:65-71 (1994)).
The invention also encompasses ribozymes, which are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. The level of expression of the RBM15-MKL1, MKL1-RBM15S, and/or MKL1-RBM15S+AE fusion proteins can also be controlled through the use of ribozyme technology. Ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)) can be used to catalytically cleave RBM15-MKL1, MKL1-RBM1S, or MKL1-RBM15S+AE mRNA transcripts to thereby inhibit translation of RBM15-MKL1, MKL1-RBM15S, and MKL1-RBM15S+AE mRNA, respectively. A ribozyme having specificity for an RBM15-MKL1-, MKL1-RBM15S-, or MKL1-RBM15S+AE-encoding nucleic acid can be designed based upon the nucleotide sequence of the corresponding cDNA disclosed herein (e.g., SEQ ID NOS: 1, 3, and 5, respectively). See, e.g., Cech et al., U.S. Pat. No. 4,987,071; and Cech et al., U.S. Pat. No. 5,116,742. Alternatively, RBM15-MKL1, MKL1-RBM15S, or MKL1-RBM15S+AE mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel and Szostak (1993) Science 261:1411-1418.
The present invention further provides methods of generating transgenic animals and transformed cell lines which contain the RBM15-MKL1, MKL1-RBM15S, and/or MKL1-RBM15S+AE nucleotide sequences. Such animals and cell lines are useful as animal models for human t(1;22) leukemias. In general, methods of generating transgenic animals and transformed cell lines are well known in the art (for example, see Grosveld et al., Transgenic Animals, Academic Press Ltd., San Diego, Calif. (1992)). Using the nucleotide sequences disclosed herein for the RBM15-MKL1, MKL1-RBM15S, or MKL1-RBM15S+AE or coding sequences for the RBM15-MKL1, MKL1-RBM15S, and/or MKL1-RBM15S+AE fusion proteins, a skilled artisan can readily generate a transgenic animal and transformed cell lines which contains and expresses the RBM15-MKL1 fusion protein, the MKL1-RBM15S fusion protein, and/or MKL1-RBM15S+AE fusion protein. Transgenic animals (such as mice and pigs) which express the RBM15-MKL1 fusion gene can be used as an animal model for human t(1;22) leukemia. Transgenic animals which express the RBM15-MKL1 fusion protein, the MKL1-RBM15S fusion protein, or MKL1-RBM15S+AE fusion protein, or any combination thereof, are useful for determining, at the molecular level, the roles of the RBM15-MKL1, MKL1-RBM15S, and MKL1-RBM15S+AE fusion proteins in the development of acute megakaryoblastic leukemia. Such animals serve as models for the development of alternative therapies for t(1;22) lymphoma.
Transformed eukaryotic cell lines that express on or more of the fusion of proteins of the invention can be used, for example, to screen for agents that are useful for treating AMKL. Preferably such cell lines are mammalian cell lines. More preferably, such cell lines are human cell lines. Generally, desired agents are those that suppress or eliminate phenotypic changes that occur as a result of the expression of one or more fusion proteins of the invention in the cell. Phenotypic changes that occur as a result of the expression of one or more fusion proteins of the invention in the cell include, for example, the presence of CD61 and absence of peroxidase and esterase (See, e.g. Bennett et al., Ann. Intern Med 103:460-462 (1985); Skinnider L. F. et al., Acta Haematologica 98 (1): 26 (1997); Avanzi et al., J. Cell Physiol. 145: 458-464 (1990); Avanzi et al., Brit. J Haematol. 69: 359 (1988)).
Such desired agents may be further screened for selectivity by determining whether they suppress or eliminate phenotyic changes or activities associated with expression of unfused RBM15 and/or MKL1 proteins in cells that either express such unfused proteins naturally or are engineered to express such proteins. Selective agents are those which suppress or eliminate phenotypes associated with expression of the fusion protein but which do not suppress or eliminate the phenotypes associated with the unfused RBM15 and MKL1 proteins. Typically, the agents are screened by administering the agent to the cell. It is recognized that it is preferable that a range of dosages of a particular agent be administered to the cells to determine if the agent is useful for treating AMKL. Appropriate cell lines that can be used in this method include, but are not limited to, DAMI, MEG-01, M-07e, CMK, CHRF-288-11 and UT7 cells. In another embodiment of the present invention, methods are provided for identifying agents which are capable of binding to the RBM15-MKL1, MKL1-RBM15S, or MKL1-RBM15S+AE fusion proteins herein described. Such methods comprise (a) contacting a candidate agent with RBM15-MKL1, MKL1-RBM15S, or MKL1-RBM15S+AE fusion protein, or fragment thereof, and (b) determining whether the candidate agent binds to the fusion protein. Using this method, agents which can be used to modulate the activity of the RBM15-MKL1, MKL1-RBM15S, or MKL1-RBM15S+AE fusion protein can be identified. Such methods can additionally comprise an additional step to select from the identified agents those which do not bind RBM15 or MKL1 proteins. Such an additional step involves contacting the agent with an RBM15 or MKL1 protein, or fragment thereof and determining whether the agent binds to the protein or fragment.
There are numerous variations of the above assays which can be used by a skilled artisan without the need for undue experimentation in order to isolate agonists, antagonists, and ligands of the RBM15-MKL1, MKL1-RBM15S, and/or MKL1-RBM15S+AE fusion protein; see, for example, Burch, R. M., in Medications Development. Drug Discovery, Databases, and Computer-Aided Drug Design, NIDA Research Monograph 134, NIH Publication No. 93-3638, Rapaka, R. S., and Hawks, R. L., eds., U.S. Dept. of Health and Human Services, Rockville, Md. (1993), pages 37-45. For example, an idiotypic antibody to RBM15-MKL1, MKL1-RBM15S, or MKL1-RBM15S+AE fusion protein can be used to co-precipitate fusion protein-bound agents in the purification and characterization of such agents. Harlow, E., et al., Chapter 11 in Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring harbor, N.Y. (1988), pages 421-470. Further, an anti-idiotypic antibody to RBM15-MKL1, MKL1-RBM15S, or MKL1-RBM15S+AE can be used to design synthetic RBM15-MKL1, MKL1-RBM15S, or MKL1-RBM15S+AE ligands. Ertl, H., et al., Vaccine 6:80-84 (1988); Wolff, M. E., in Medications Development: Drug Discovery, Databases, and Computer-Aided Drug Design, NIDA Research Monograph 134, NIH Publication No. 93-3638, Rapaka, R. S., and Hawks, R. L., eds., U.S. Dept. of Health and Human Services, Rockville, Md. (1993), pages 46-57. In addition, an anti-idiotypic antibody to the RBM15-MKL1, MKL1-RBM15S, or MKL1-RBM15S+AE fusion proteins, the RBM15-MKL1, MKL1-RBM15S, or MKL1-RBM15S+AE fusion proteins, or a fragment thereof containing the active (ligand binding) site of the fusion protein, can be used to screen an expression library for genes encoding proteins which bind the fusion protein.
Alternatively, cells expressing the RBM15-MKL1, MKL1-RBM15S, or MKL1-RBM15S+AE fusion proteins on their surfaces can be used to screen expression libraries or synthetic combinatorial oligopeptide libraries. Cwirla, S. E., et al., Proc. Natl. Acad. Sci. (USA) 87:6378-6382 (1990); Houghten, R. A., et al., Nature 354:84-86 (1991); Houghten, R. A., et al., in Medications Development: Drug Discovery, Databases, and Computer-Aided Drug Design, NIDA Research Monograph 134, NIH Publication No. 93-3638, Rapaka, R. S., and Hawks, R. L., eds., U.S. Dept. of Health and Human Services, Rockville, Md. (1993), pages 66-74. In particular, cells that have been genetically engineered to express and display the RBM15-MKL1, MKL1-RBM15S, and/or MKL1-RBM15S+AE fusion protein via the use of the nucleic sequences of the invention are preferred in such methods, as host cell lines may be chosen which are devoid of related receptors. Hartig, P. R., in Medications Development: Drug Discovery, Databases, and Computer-Aided Drug Design, NIDA Research Monograph 134, NIH Publication No. 93-3638, Rapaka, R. S., and Hawks, R. L., eds., U.S. Dept. of Health and Human Services, Rockville, Md. (1993), pages 58-65.
The agents screened in the above assay can be, but are not limited to, small molecules, peptides, carbohydrates, or vitamin derivatives. The agents can be selected and screened at random or rationally selected or designed using protein modeling techniques. For random screening, agents such as peptides or carbohydrates are selected at random and are assayed for their ability to bind to the pseudogene peptide. Alternatively, agents may be rationally selected or designed. As used herein, an agent is said to be “rationally selected or designed” when the agent is chosen based on the configuration of the pseudogene peptide. For example, one skilled in the art can readily adapt currently available procedures to generate peptides capable of binding to a specific peptide sequence in order to generate rationally designed antipeptide peptides, see, for example, Hurby et al., “Application of Synthetic Peptides: Antisense Peptides,” in Synthetic Peptides: A User's Guide, W. H. Freeman, New York (1992), pp. 289-307; and Kaspczak et al., Biochemistry 28:9230-2938 (1989).
Using the above procedures, the present invention provides agents capable of binding to the the RBM15-MKL1, MKL1-RBM15S, and/or MKL1-RBM15S+AE fusion proteins, produced by a method comprising the steps of (a) contacting said agent with the the RBM15-MKL1, MKL1-RBM15S, and/or MKL1-RBM15S+AE fusion protein, or a fragment thereof, and (b) determining whether said agent binds to the RBM15-MKL1, MKL1-RBM15S, or MKL1-RBM15S+AE fusion protein. Additional step(s) to determine whether such binding is selective for the fusion protein relative to the corresponding unfused RBM15 and MKL1 proteins may also be employed.
The materials used in the above assay methods (both nucleic acid and protein based) are ideally suited for the preparation of a kit. For example, for amplification based detection systems, the invention provides a compartmentalized kit to receive in close confinement, one or more containers which comprises (a) a first container comprising one or more of the amplification primers of the present invention, and (b) one or more other containers comprising one or more of the following: a sample reservoir, amplification reagents, wash reagents, and detection reagents.
For antibody based detection systems, the present invention provides a compartmentalized kit to receive in close confinement, one or more containers which comprises (a) a first container comprising an antibody capable of binding to the RBM15-MKL1, MKL1-RBM15S, or MKL1-RBM15S+AE fusion protein and (b) one or more other containers comprising one or more of the following: wash reagents and reagents capable of detecting the presence of bound antibodies from the first and the second containers.
The invention further provides a kit compartmentalized to receive in close confinement one or more containers which comprises (a) a first container comprising an antibody capable of binding to an epitope which is present in the fusion junction of the RBM15-MKL1, MKL1-RBM15S, or MKL1-RBM15S+AE fusion protein and which is not present in either of the two non-fusion proteins; and (b) one or more other containers comprising one or more of the following: wash reagents and reagents capable of detecting the presence of bound antibodies from the first container.
In detail, a compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper. Such containers allow one to efficiently transfer reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated, and the agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers may include a container which will accept the test sample, a container which contains the antibodies or probes used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, etc.), and containers which contain the reagents used to detect the bound antibody or the hybridized probe. Any detection reagents known in the art can be used including, but not limited to those described supra.
The following examples are offered by way of illustration and not by way of limitation.
EXPERIMENTAL
EXAMPLE 1
Fusion of RNA Recognition Motif-Encoding Gene, RBM15, to the SAP DNA-Binding Domain Gene, MegaKaryoblastic Leukemia-1 (MKL1), in Acute Megakaryoblastic Leukemias with t(1;22)(p13;q13)
Summary
Acute megakaryoblastic leukemia (AMKL) in young children is almost invariably caused by leukemic blasts harboring t(1;22)(p13;q13) (Carroll, A. et al., Blood 78:748-752 (1991); Lion, T. et al., Blood 79:3325-3330 (1992); Bernstein, J. et al., Leukemia 14:216-218 (2000)). Despite its remarkable disease specificity and a lack of knowledge of AMKL pathogenesis (Cripe, L. D. & Hromas, R., Semin. Hematol. 35:200-209 (1998)), t(1;22) has yet to be characterized molecularly. Disclosed herein is the identification of the reciprocal fusion transcripts derived from two novel genes, RNA-binding motif protein-15 (RBM15) at chromosome 1p13 and Megakaryoblastic Leukemia-1 (MKL1) at 22q13, as the consequence of t(1;22). RBM15, detected in three isoforms—RBM15L, RBM15S, and RBM15S+AE—contains three RNA recognition motifs (RRM) (Burd, C. G. & Dreyfuss, G., Science 265:615-621 (1994)) and a Spen paralog and ortholog C-terminal (SPOC) domain (Wiellette, E. L. et al., Development 126:5373-5385 (1999)), thus showing significant homology to spen, a homeotic Drosophila gene capable of enhancing Ras/MAP kinase signaling (Wiellette, E. L. et al., Development 126:5373-5385 (1999); Rebay, I. et al., Genetics 154:695-712 (2000); Chen, F. & Rebay, I., Curr. Biol. 10:943-946 (2000); Kuang, B. et al., Development 127:1517-1529 (2000)). MKL1 contains a SAP (SAF-A/B, Acinus and PIAS) DNA-binding motif (Aravind, L. & Koonin, E. V., Trends Biochem. Sci. 25:112-114 (2000)) that in homologous proteins such as SAF-B functions to recruit domains involved in chromatin remodeling, transcriptional control, and pre-mRNA processing to the matrix attachment regions (MAR) of transcriptionally active chromatin, effectively coupling transcription and splicing (Naylor, O. et al., Nucleic Acids Res. 26:3542-3549 (1998)). Although both reciprocal fusion transcripts are expressed in AMKL, RBM15-MKL1, from the der(22) chromosome, encodes all putative functional motifs of each gene and is the candidate oncogene of t(1;22), through a mechanism that may involve deregulation of RNA processing and/or Hox and Ras/MAP kinase signaling.
Description
Cloning of chromosomal translocations has led to identification of pathogenically relevant oncogenic fusion transcripts and proteins in specific subsets of acute nonlymphocytic leukemia (ANLL), such as promyelocytic leukemia-retinoic acid receptor alpha fusion gene (PML-RARα) in acute promyelocytic leukemia (FAB-M3 subtype), acute myeloid leukemia 1-eight twenty one fusion gene (AML1-ETO) in ANLL with maturation (FAB-M2), and various mixed lineage leukemia (MLL) gene fusions in acute myelomonocytic and monocytic leukemias (FAB-M4 and -M5) (Melnick, A. & Licht, J. D., Blood 93:3167-3215 (1999); Downing, J. R., Br. J. Haematol. 106:296-308 (1999); Rowley, J. D., Semin. Hematol. 36:59-72 (1999); Look, A. T., Science 278:1059-1064 (1997); Faretta, M., Di Croce, L. & Pelicci, P. G., Sem. Hematol. 38:42-53 (2001)). Despite these significant advances, little is known about the genetic mechanisms underlying acute leukemias of the megakaryoblastic (platelet precursor) lineage (AMKL, FAB-M7) (Cripe, L. D, infra). Almost invariably, AMKL in non-Down syndrome infants and young children harbor the t(1;22)(p13;q13), in most cases as the sole cytogenetic abnormality(Carroll, A. et al; Lion, T. et al., and Bernstein, J. et al., infra). Phenotypically, AMKL presents de novo (i.e., without a so-called preleukemic stage), with a large leukemia cell mass, and frequent fibrosis of bone marrow and other organs. Progression is usually rapid despite therapy, with a median overall patient survival of only 8 months.
To clone t(1;22), a fluorescence in situ hybridization (FISH)-based positional cloning strategy was used to define the 1p13 and 22q13 breakpoints . Bacterial artificial chromosome (BAC) clones mapping to each chromosomal band were selected using the public and Celera human sequence databases and used pairwise in a series of two-color, two-probe FISH analyses of metaphase chromosomes from t(1;22)-containing leukemia blasts to identify closely flanking clones. With this strategy, a single chromosome 22 BAC clone, RP5-1042K10, was found that hybridized to both the der(1) and der(22) chromosomes formed by the reciprocal balanced t(1;22) and thus contained the altered 22q13 gene locus. Additional FISH experiments using DNA subfragments of RP5-1042K10 prepared by long-distance PCR (LD-PCR) methods allowed refinement of the chromosome 22 breakpoint to within the most telomeric gene in this clone, and revealed that all cases with t(1;22) possessed genomic breakpoints in a single  ̃28-kb intron of this gene. The same approach demonstrated the breakpoint on chromosome 1p13 to be encompassed by BAC clone RP 11-260A24. After complete annotation of the sequence by combining entries in the public and Celera databases, the 1p13 breakpoint was sublocalized to within an  ̃6-kb genomic interval (extending from nucleotide 1,798,871 to 1,804,858 in Celera scaffold GA_x2HTBL4WN8M) using RP11-260A24 LD-PCR subfragments in metaphase FISH. FISH analyses with probes closely flanking the identified breakpoint regions on chromosome 1 and chromosome 22 confirmed the results of our ‘split signal’ analysis, highlighting both the der(1) and der(22) chromosomes. The breakpoint-spanning clones from each chromosome identified by FISH analysis are RP11-260A24 (chromosome 1) and RP5-1042K10 (chromosome 22). An additional 22q13 BAC, clone RP11-313L7, that spanned the breakpoint was subsequently also identified, the end sequences (accession nos. AQ506839 (Sp6) and AQ537696 (T7)) of which revealed a 65,460 bp overlap with RP5-1042K10 and a 76,650 bp overlap with an additional chromosome 22 clone for this region designated RP4-591N18.
Database searches using the exon sequences flanking the breakpoint-containing intron on chromosome 22 identified an anonymous human brain cDNA library clone (accession no. AB037859). This 3,907-bp cDNA encoded a 2,793-nucleotide ORF, with the putative ATG initiator codon in a context (ATCatgC) adequate to support translational initiation (Kozak, M., Mammalian Genome 7:563-574 (1996)). Because RNA blot hybridizations using this clone revealed an approximately 4.5-kb transcript expressed ubiquitously in normal human tissues, additional 5′ untranslated sequence was obtained by RACE, resulting in a complete cDNA of 4,447 bp (SEQ ID NO: 13). To denote its involvement in AMKL, this gene was named MKL1 (Megakaryoblastic Leukemia-1, official HUGO Nomenclature Committee designation). Motif searches of the deduced 931-amino acid (aa) MKL1 protein (predicted mass, 98.9 kDa; SEQ ID NO: 14) identified a bipartite nuclear localization signal (BP-NLS) (residues 14-31 of SEQ ID NO: 14; RRSLERARTEDYLKRKIR), a single SAP DNA-binding motif (Aravind, L. & Koonin, E. V., Trends Biochem. Sci. 25, 112-114 (2000)) (residues 347-381 of SEQ ID NO: 14), a coiled-coil region (residues 521-563 of SEQ ID NO: 14) that likely mediates protein oligomerization (Lupas, A., Trends Biochem. Sci. 21, 375-382 (1996)), and a long C-terminal proline-rich segment (residues 564-811 of SEQ ID NO: 14) similar to proline-rich regions shown to act as transcriptional activators (Mitchell, P. J. & Tjian, R., Science 245, 372-378 (1989)). In addition, a short glutamine-rich segment (residues 264-286 of SEQ ID NO: 14; QQQQLFLQLQILNQQQQQHHNYQ) was found that is highly reminiscent of the more extensive glutamine-rich regions of the MLL acute leukemia-associated transcription factor family, as well as a number of other proteins involved in transcriptional control (Prasad, R. et al., Oncogene 15, 549-560 (1997)). Of note, MKL1 showed significant cross-species homology to the product of a Drosophila gene of undetermined function, CG12188 (accession no. AAF47681), exhibiting 41% identity (57% similarity) over the initial 161 amino acids of MKL1 and 63% identity (76% similarity) in the SAP domains of the two proteins.
The MKL1 SAP domain shares sequence similarities with SAP domains from (a) THO1—yeast protein Tho1p, which regulates transcriptional elongation by RNA polymerase II; (b) E1B-55 kDa, a transforming adenovirus protein that binds and inhibits p53, and mediates nucleocytoplasmic transport of adenoviral and cellular mRNAs; (c) PIAS1 (protein inhibitor of activated Stat1) which binds and inhibits Stat1, coactivates transcription by various steroid receptors, regulates RNA helicase II function, and has also been reported to bind wt and mutant p53; (d) SAF-B (scaffold attachment factor B), a RRM-containing protein that binds both RNA polymerase II and a subset of serine-/arginine-rich RNA splicing factors; and (e) ACINUS (apoptotic chromatin condensation inducer in the nucleus) which mediates chromatin condensation during programmed cell death (reviewed in Aravind, L. & Koonin, E. V., Trends Biochem. Sci. 25, 112-114 (2000)).
With the hypothesis that t(1;22) generates an oncogenic fusion analogous to breakpoint cluster region-Abelson tyrosine kinase fusion gene (BCR-ABL) or the Mixed-lineage leukemia (MLL)fusion genes in leukemias, 5′ RACE was performed with total RNA from our leukemia patient samples using MKL1 oligonucleotide primers to identify the 1p13 fusion partner. The obtained sequences corresponded to two anonymous, partially overlapping cDNA clones (accession nos. AK025596, AK022541) from chromosome 1 that were also contained within the approximately 6-kb genomic interval in BAC RP11-260A24 previously demonstrated by FISH to span the 1p13 breakpoint. Due to the presence of three RNA recognition motifs (RRM) encoded by these sequences, the corresponding gene was named RBM15 (RNA-binding motif protein-15, official HUGO Nomenclature Committee name). Using 5′ RACE and human placenta cDNA library screening, the complete RBM15 coding sequence was obtained by identification of an ATG initiator codon 132 nucleotides (nts) upstream of the previously deposited sequences and preceded 30 nts by an in-frame TGA stop. Sequencing of RT-PCR products obtained with RBM15-specific primers from normal leukocyte mRNA demonstrated three transcripts that share an identical 2,863-bp 5′ coding sequence, differing only in their extreme 3′ coding portions due to alternative exon usage (FIG. 1)
RBM15 contains three RRM motifs and a BP-NLS in its C-terminus, and is highly homologous to Drosophila gene product GH11110 (accession no. AF 145664) (40% identity, 56% similarity with the RRM-containing region of RBM15 from residues 170-529; 39% identity, 54% similarity with the RBM15 C-terminus from residues 714-954; percent identity determined using BLAST 2.1.3 (Altschul, S. F. et al., Nucleic Acids Res. 25: 3389 (1997)) with default parameters selected). These regions of RBM15 and Drosophila GH11110 (also previously referred to as D. melanogaster Short spen-like protein-2, DmSSLP2) (Wiellette, E. L. et al., Development 126, 5373-5385 (1999)) are closely related to Drosophila spen (split ends)—an RRM protein that modulates Hox homeotic function (e.g., cooperating with Antennapedia to suppress head-like development in the thoracic region), and regulates neuronal cell fate and axon extension by enhancing Ras/MAP kinase signaling (Wiellette, E. L. et al., Development 126, 5373-5385 (1999); Rebay, I. et al., Genetics 154, 695-712 (2000); Chen, F. & Rebay, I., Curr. Biol. 10, 943-946 (2000); Kuang, B. et al., Development 127, 1517-1529 (2000)). Thus, the RBM15 C-terminus also contains a so-called SPOC (Spen paralog and ortholog C-terminal) domain, a 165-aa conserved motif of undetermined function found in Spen and Spen-like proteins (Wiellette, E. L. et al., Development 126, 5373-5385 (1999)) including the mammalian spen ortholog, MINT (Msx2-interacting nuclear target), which binds homeoprotein Msx2 (Hox 8) and coregulates osteoblast gene expression during craniofacial development (Newberry, E. P. et al., Biochemistry 38, 10678-10690 (1999)).
In t(1;22)-positive AMKL blasts, RT-PCR demonstrated expression of both reciprocal fusion transcripts, RBM15-MKL1 and MKL1-RBM15. RT-PCR reactions using RBM15 sense (RBM15(S)-2746F) and MKL1 antisense (MKL1-204R) primers amplify a single 268-bp RBM15-MKL1 product in all patients (nucleotides 2866 to 3133 of SEQ ID NO: 1). Using MKL1 sense (MKL1-F) and RBM15 antisense (RBM15(S)-2930R, corresponding to sequences of the 3′ most exon unique to RBM15S and RBM15S+AE) primers, two reciprocal MKL1-RBM15 fusion transcripts are detected, one (251 bp; nucleotides 411 to 661 of SEQ ID NO: 3) containing the 3′ sequences from RBM15S and the other (362 bp; nucleotides 411 to 772 of SEQ ID NO: 5) with 3′ sequences found in RBM15S+AE . No MKL1-RBM15 RT-PCR products were obtained using MKL1-F and an antisense primer specific for RBM15L (RBM15(L)-1636R) in any patients examined.
The predicted RBM15-MKL1 chimeric protein encoded on the der(22) contains all putative functional motifs of each normal protein (FIG. 1). Frequent duplication of der(1) in t(1;22)-containing blasts has led to speculation that this abnormal chromosome likely encodes the oncogenic AMKL fusion protein(s) (Carroll, A. et al.; Lion, T. et al., Bernstein, J. et al., infra.); however, a functional role for MKL1-RBM15S and MKL1-RBM15S+AE in leukemogenesis is unclear given they encode predicted products of only 17 and 25 aa, respectively.
The SAP motif mediates DNA binding of proteins to the AT-rich matrix attachment regions (MAR) associated with transcriptionally active chromatin (Aravind, L. & Koonin, E. V., Trends Biochem. Sci. 25, 112-114 (2000)). SAP proteins include SAF-B (Chen, F. & Rebay, I., Curr. Biol. 10, 943-946 (2000)), involved in RNA processing; Acinus (Sahara, S. et al., Nature 401, 168-173 (1999)), which induces chromatin condensation; and PIAS proteins (Valdez, B. C. et al., Biochem. Biophys. Res. Commun. 234, 335-340 (1997); Chung, C. D. et al., Science 278, 1803-1805 (1997); Kotaja, N. et al., Mol. Endocrinol. 14, 1986-2000 (2000)) that bind RNA helicase II, inhibit STAT signal transduction, and modulate steroid receptor-dependent transcription. Thus, the SAP targets a diverse set of functional domains to MAR sequences, coupling transcription and splicing. In addition to modulating homeotic protein functions (and in the case of spen, enhancing Ras/MAP kinase signals), Spen family proteins like MINT can bind specific DNA sequences via their RRM domains, an RRM function seen also in other transcriptional regulators such as sea urchin SSAP (Stage-specific activator protein) (DeAngelo, D. J. et al., Mol. Cell. Biol. 15, 1254-1264 (1995); DeFalco, J. & Childs, G., Proc. Natl. Acad. Sci. 93, 5802-5807 (1996)) and hTAFII68, an RNA/ssDNA-binding protein homologous to pro-oncoproteins TLS/FUS and EWS (Bertolotti, A. et al., EMBO J 15, 5022-5031 (1996); Bertolotti, A. et al., Oncogene 18, 8000-8010 (1999)). In RBM15-MKL1, the MKL1 SAP motif would be expected to relocalize the RRM domains of RBM15 aberrantly to sites of transcriptionally active chromatin, targeting genes critical for the normal proliferation or differentiation of megakaryoblasts.
Methods
Clinical Cases
Leukemia specimens with histopathological and immunophenotypic features typical of AMKL were studied from five infants and young children. All cases contained t(1;22)(p13;q13) with the exception of patient 2, whose blasts possessed a complex t(1;6;22)(p13;p12;q13). All five specimens were shown to contain rearrangement of RBM15 and MKL1 by RT-PCR and/or FISH analysis.
Fluorescence In Situ Hybridization (FISH)
DNA was labeled by nick translation with digoxigenin-11-dUTP and/or biotin-16-dUTP (Roche Molecular Biochemicals). Labeled probes were combined with sheared human DNA and hybridized to fixed interphase nuclei and metaphase cells in 50% formamide, 10% dextran sulfate and 2×SSC at 37° C. and subsequently washed in a 50% formamide, 2×SSC solution at 37° C. Hybridization signals were detected with fluorescein-labeled anti-digoxigenin (Ventana Medical Systems) for digoxigenin-labeled probes and Texas red-avidin for biotinylated probes. Chromosomes and nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) prior to analysis.
Rapid Amplification of cDNA Ends (RACE) and DNA Sequencing
Total RNA was extracted from t(1;22)-positive frozen AMKL specimens using RNA STAT-60 (Tel-Test, Inc.). Approximately 0.2 μg RNA was used for 5′ RACE experiments that identified RBM15 as the chromosome 1p13 partner gene of MKL1. Reverse transcription was performed with primer MKL1-294R (SEQ ID NO: 15). After purification and tailing of the cDNA, PCR was performed with an oligo-dT anchor primer and MKL1 reverse primer MKL1-73R (SEQ ID NO: 16), using temperature cycling conditions recommended by the manufacturer (Roche Molecular Biochemicals) and a Thermal Cycle Model 2400 (Perkin-Elmer Cetus). Twenty microliters of PCR product were separated on a 2% agarose gel, and specific bands were purified (Qiaquick gel extraction kit, Qiagen, Inc.) and sequenced using MKL1 reverse primer MKL1-59R (SEQ ID NO: 17).
RT-PCR of RBM15-MKL1 and MKL1-RBM15 Fusion Transcripts
For RBM15-MKL1 detection, 1 μg of total RNA was reverse transcribed using primer MKL1-294R (SEQ ID NO: 15). PCR was performed using primers RBM15(S)-2746F (SEQ ID NO: 18) and MKL1-204R (SEQ ID NO: 19) and 35 cycles (94° C. for 15 s, 60° C. for 30 s, 72° C. for 30 s). Primer pair MKL1-F (SEQ ID NO: 20) and MKL1-204R, designed to amplify a portion of the ubiquitously expressed MKL1, were included in control experiments to verify RNA quality and RT-PCR technique. PCR products were gel purified, then cycle sequenced using primers RBM15(S)-2746F and MKL1-204R. For detection of MKL1-RBM15 fusion transcripts, reverse transcription was done using an oligo-dT primer and PCR performed with primers MKL1-F (SEQ ID NO: 20) and RBM15(S)-2930R (SEQ ID NO: 21) (to identify MKL1-RBM15S and MKL1-RBM15S+AE) or MKL1-F (SEQ ID NO: 20) and RBM15(L)-1636R (SEQ ID NO: 22) (to detect MKL1-RBM15L). Amplification of normal RBM15 sequences for quality control was performed with primer pairs RBM15(S)-2746F (SEQ ID NO: 18) and RBM15(S)-2930R (SEQ ID NO: 21) or RBM15(L)-1636R (SEQ ID NO: 22).
Northern Blot Analysis
Normal human peripheral blood leukocyte total RNA was extracted (RNEasy kit, Qiagen), then treated with RNase-free DNase for 15 m at room temp. This RNA was reverse transcribed and used as the template in PCR amplifications (35 cycles: 94° C. for 10 s, 62° C. for 10 s, 68° C. for 1 m) to generate cDNA fragments corresponding to probes a-d (FIG. 1). The following PCR primer pairs were used: RBM15 probe a (433 bp), RBM15-1118F (SEQ ID NO: 23) and RBM15-1551R (SEQ ID NO: 24); RBM15 probe b (318 bp), RBM15-2831F (SEQ ID NO: 25) and RBM15-3149R (SEQ ID NO: 26); RBM15 probe c (388 bp), RBM15-1616F (SEQ ID NO: 27) and RBM15-2004R (SEQ ID NO: 28); MKL1 probe d (449 bp), MKL1-155F (SEQ ID NO: 29) and MKL1-294R (SEQ ID NO: 15). Multiple tissue Northern blots (Clontech) containing approximately 2 μg of poly(A)+RNA prepared from normal human tissues were hybridized at 68° C. for 2 h in ExpressHyb buffer (Clontech) using these RBM15 and MKL1 cDNA probes or a β-actin probe supplied by the manufacturer. Filters were autoradiographed at −80° C. with one intensifying screen for 3 d (probes a, c and d), 1 d (probe b) or 1 h (for β-actin).
All publications and patent applications mentioned in the specification are indicative of the level of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the following embodiments.

## Claims

1. An isolated nucleotide molecule comprising a nucleotide sequence selected from the group consisting of:
(a) a nucleotide sequence encoding the fusion protein having the amino acid sequence set forth in SEQ ID NO: 2; and
(b) the nucleotide sequence set forth in SEQ ID NO: 1.